Project Summary Breast cancer is a major malignancy and a leading cause of death among women worldwide. It is very well known that breast cancer is a heterogeneous disease with several major subtypes with distinct biological, molecular and clinical characteristics. One of the subtypes known as triple negative breast cancer is characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and lack of overexpression of human epidermal growth factor receptor 2 (HER2). This subtype of breast cancer occurs in 10-25% of cases and is typically associated with aggressive tumors and poor prognosis. Due to the lack of the common receptors, these types of tumors do not respond to receptor-targeted therapies, and even the chemotherapy is not very effective in this type of breast cancer. Hence, there is an urgent need to find alternative effective therapeutics for the treatment of TNBC patients. In this application, we propose to study the role of a potential secreted peptide called Humanin (HN), which could be targeted for TNBC treatment. At present, nothing is known about the novel role of this peptide in breast cancer. HN was originally discovered as a strong neuro-protective anti-apoptotic secreted peptide. Interestingly, it is encoded by mitochondrial DNA inside a 16s rRNA gene. Our preliminary data support the potential role of this novel peptide in breast cancer, in particular, TNBC. Here we plan to build upon our preliminary findings and define the role of HN in TNBC. We also propose proof of principle studies to determine whether blocking this peptide could help in the treatment of TNBC. The specific aims of the proposal are- Aim 1: Determine whether Humanin (HN) is overexpressed in breast cancer cell lines and breast cancer specimens, in particular, triple negative breast cancers (TNBC). Aim 2: Examine the role of HN in promoting breast cancer stem cell (BCSC) phenotype. Aim 3: Determine the role of Humanin in breast cancer progression and examine whether blocking HN can inhibit breast cancer growth. The mechanistic studies related to the role of HN in breast cancer progression will help develop therapies that target HN and its downstream pathways to inhibit breast cancer cell proliferation and induce cell death to treat TNBC. In summary, the proposed studies will test a novel hypothesis that a mitochondrially encoded secreted peptide may be a key player in the development of TNBC, and that this secreted peptide could be targeted for the therapy of TNBC tumors, which are the most aggressive, and at present most difficult to treat using conventional receptor-targeted therapies.